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A novel HPLC/UV method for the determination of obeticholic acid and its impurities was presented.Related compounds of obeticholic acid were classified into three group based on the mass spectra.The intense adduct ions of phosphoric acid ([M + H + H3PO4]+ and [M + H + 2H3PO4]+) were described.The proposed HPLC/UV method was validated in accordance with ICH guidelines.An HPLC method with UV and electrospray ionization – mass spectrometry (ESI–MS) detection was developed for the separation and determination of obeticholic acid (OBE) and its related compounds in development process and quality control. OBE and its related compounds were classified into three major group based on the mass spectra profiles: (A) those containing a hydroxyl group at position 3 and 7, (B) those containing a hydroxyl group and/or carbonyl group at position 3, hydrogen, ethyl or ethylidene group at position 6 and a hydroxyl group and/or carbonyl group at position 7, and (C) those containing carbonyl groups at position 3 and 7. ESI–MS ionization of OBE and its related compounds often produced intense adduct ions [M + H + 98]+ and/or [M + H + 196]+ that were identified as the adduct ions of phosphoric acid ([M + H + H3PO4]+ and [M + H + 2H3PO4]+) originating from the mobile phase. The separation on HPLC system was accomplished using stationary phase based on XSelect CSH C18 (3.0 × 150 mm × 2.5 μm) and a linear gradient elution using acetonitrile and 0.05% of o-phosphoric acid. The condition of chromatographic system was set as follows: flow rate 0.7 mL/min, temperature 45 °C and UV detection at 192 nm. The separation of the 19 compounds was finished in less than 18 min (including equilibration time). The HPLC/UV method was partially validated according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines in terms of repeatability, selectivity, linearity and limit of quantification and detection.